Scientific paper summary

Molecular phylogenetic analysis of Bacillus symbionts of bees fossilized 23-40 million years ago in amber.

Cano, R.J., Borucki, M.K., Higby-Schweitzer, M., Poiner, H.N., Poiner, G.O., and Pollard, K.J. 1994 Bacillus DNA in fossil bees: An ancient symbiosis? Appl. Env. Microbiol. 60:2164-2167.

Question : Can DNA, and the information it contains, be extracted from bacteria trapped in fossilized sap?

Bees typically have intimate symbioses with Bacteria of the genus Bacillus. Bacillus can be be found not only in the gut, but also the trachea and blood (called hemolymph in arthropods) of healthy animals. The symbiosis is established when they emerge as adults from pupae and begin to feed on pollen. The Bacteria are involved in the digestion of pollen and in disease prevention, apparently by the production of antibiotics (rememeber that insects do not have the adaptive immunity systems found in vertebrates).

In this paper, by Cano, et al., the isolation of DNA and PCR amplification of bacterial ssu rRNAs from the abdomens of 23-40 million-year-old amberized bees is described. Can you say "Jurassic Park"?

The bees were trapped in pine sap during the Lower Miocene to Upper Eocene periods, and the sap was fossilized as amber. The authors obtained 11 amberized bees of the species Propeneia dominicana, an extinct stingless bee. The surface of the amber was sterilized, and then opened aseptically. The head, thorax and abdomens were separated and DNA was isolated from each. The rational here is that in living bees, Bacillus is found predominantly or exclusively in the abdomens, and the species selected was chosen so that they could be compared to living bees that are apparently closely related.

6 of the 11 bees yeilded PCR products of the correct size, and when these were sequenced and analyzed phylogenetically, turned out to be from the genus Bacillus. Only samples from the abdomen yeilded PCR products. The authors conclude that they have isolated DNA from Bacillus (probably from spores) that grew in the bees millions of years ago, and that the bee-Bacillus symbiosis has been a part of the life cycle of these organisms for at least 25-40 million years.

There are lots of similar reports of DNA sequences from the animals (and plants) trapped in more recent amber or sedimentary deposits. For example, one of the earliest such reports was of ribulose-bis-phosphate carboxylase-oxygenease sequences obtained from an 11 million-year-old magnolia leaf. As far as I know, no-one has obtained any sequences old enough to be contemporary with dinosaurs, which became extinct (ignoring birds) about 65 million years ago.

After this paper, the authors went ahead with the obvious experiment - trying to cultivate Bacillus from similar amberized bee samples. The authors went to extraordinary lengths to preclude contamination from the outside, drilling into the surface-sterilized amber and sucking out the innards of the bees aseptically. And they were able to isolate Bacillus from these amberized bees! Still, many do not believe that the Bacillus isolates are ancient, but probably contamination. One of the arguments is that the organisms isolated (originally a species of Bacillus sphaericus) are really very closely related to modern species - and don't fall out in the tree where you might expect an ancient specie to reside. However, so many isolates have now been revived, including the apparent Bacillus strains from which the original sequences were obtained, and so many nice controls have been performed, that it's possible that these really are cultures revived from spores that have been in suspended animation for millions of years.

A more "out-there" claim is that Bacillus cultures have been revived from spores trapped in salt crystals (now halite) in a dying salt lake 250 million years ago. The experiments are great, but there is real doubt about the real age of the aqueous inclusions the spores came from, and the cultures are indistinguishable from modern Bacillus species.